Identification and characterization of novel virulence factors in Streptococcus pneumoniae

Abstract: Streptococcus pneumoniae (the pneumococcus) is a major human pathogen with high morbidity and mortality worldwide. Increased antibiotic resistance and insufficient vaccination contribute to the re-emerging of this pathogen. Identifying novel virulence factors could lead to a better understanding of the pathology of pneumococcal disease and result in novel therapeutic approaches. We were able to show the presence of a surface-exposed pilus structure in pneumococci, made up of three distinct protein subunits, namely RrgA, RrgB and RrgC, which are encoded by the rlrA islet. Piliated strains showed better adherence to lung epithelial cells in vitro and were more virulent in vivo than non-piliated strains. We assessed the protective potential of pilus proteins in a mouse model of pneumococcal pneumonia and found that intranasal immunization with RrgB protected from a lethal intranasal challenge and reduced bacterial loads in the nasopharynx, lungs and bloodstream. Hence, the pneumococcal pilus is an important virulence factor, functioning as an adhesin that might be used for vaccination against pneumococcal disease. Pneumococci contain an antiphagocytic capsule that protects them from phagocytosis by neutrophils and macrophages. Nevertheless neutrophils are abundant in pneumococcal disease. Studying the interaction of pneumococci and neutrophils in more detail, we found that neutrophil extracellular traps (NETs) were formed during pneumococcal pneumonia in a mouse model. NETs are extracellular chromatin DNA networks with embedded enzymes and cationic antimicrobial peptides. Pneumococci were trapped by NETs in vitro but, unlike many other pathogens, not killed. We could show that the surface located DNase EndA in pneumococci degraded the DNA scaffold of NETs and allowed pneumococci to escape trapping, which promoted virulence in a mouse model of pneumococcal pneumonia. Pneumococci also incorporate positively charged D-alanine residues into surface (lipo)teichoic acids, leading to the repulsion of antimicrobial peptides. We found that the inactivation of this mechanism in non-encapsulated pneumococci rendered the organism sensitive to killing by NETs. In a murine model even the encapsulated D-alanylation mutant showed decreased virulence, suggesting a role for this mechanism at the early stage of disease when expression of capsule polysaccharide has been shown to be low. We also found that encapsulation significantly reduced trapping by NETs. The combination of capsule, DNase and incorporation of positive surface charge make NETs virtually ineffective in clearing pneumococci. These findings could lead to novel therapeutic approaches in the form of DNase- or D-alanylation-inhibitors to fight pneumococcal infections.